Advances in cell separation technology have spawned therapeutic methods in which a subject's cells can be removed from the bloodstream or bone marrow and fractionated to provide specific cell types for re-introduction into the subject or of a recipient patient. For example, U.S. Pat. No. 5,840,502, filed Aug. 31, 1994, describes methods for enriching cell fractions and indications for use of such fractions.
In enriching a cell fraction from a cell suspension, it is many times desirable to add reagents, such as cell-specific antibodies or buffering agents, to the cell suspension as part of the fractionation procedure. Such reagents must be removed before re-introduction of the cells into a patient. Alternatively or in addition, it is many times desirable to change the ionic conditions of the cells prior to use of the cells, such as in the therapeutic indications referred to above.
As such cell fractionation procedures become routine in the clinical setting, it is desirable that handling and manipulation of the cells be minimized, so that cells can be processed in a minimum amount of time and with a minimum amount of exposure to potential contamination.
Typically, isolated cells are washed by either resuspending the cells in the same centrifuge tube or bag in which they were originally centrifuged or by transferring the cells to a different centrifugable container. Cells are resuspended in the wash buffer and re-centrifuged at relatively low centrifugal forces (approx. 1000.times.g). The cells form a soft pellet from which the wash supernatant must be removed prior to subsequent washings or resuspension in the final buffer.
Removal of supernatant can be effected either by decantation or by gentle aspiration. Decantation, while a relatively fast operation, often results in cell loss, differentially depleting those cells having relatively low specific gravities and which sediment on the top of the pellet. For this reason, decantation has generally not been considered to be a reliable means for removing supernatants from cells, particularly when such cells have relatively low specific gravities.
Aspiration, on the other hand, is labor-intensive, and unless careful attention is given to each individual tube, may also selectively result in loss of "lighter" cells from the pellet into the discarded washing solution. In addition, aspiration requires introduction of a probe into the cell container. This may also introduce contamination into the container.
One attempt to solve the foregoing problems is found in U.S. Pat. No. 5,047,004 (Wells) which describes an automatic decanting centrifuge in which swinging buckets are locked in an extended angle following centrifugation, to effect gravity decantation of the fluid therein. This system, while providing relative ease and automaticity to the decantation process, necessarily exposes the cells to contamination, by its open top design. Moreover, there is no provision for ensuring that the more slowly sedimenting "light" cells are retained in the pellet.
U.S. Pat. No. 5,474,687 describes a method and specialized tube for enriching an exemplified fraction of rare cells, CD34.sup.+ hematopoietic progenitor cells, in a single-step density gradient, by selectively collecting the "light" cells that migrate to the cell solution-density gradient interface. However, these cells must be pelleted and washed prior to use. Such pelleting and washing is typically carried out at relatively low speeds (500-1000.times.g) in preparative centrifuge tubes that are available commercially. Using such conventional washing methods, it has been found that the CD34.sup.+ cells are differentially lost during the washing procedure, since they are "light" cells that tend to sediment to the top of the pellet formed during the washing process.
The present invention provides a cell washing device that overcomes the problems just described. The device includes a tube with a sealable cap or lid that provides for sterile transfer and handling of cells. Specifically, cells or liquid medium can be added to the tube by means of a sterile port which traverses the cap. In addition, according to an important feature of the invention, wash supernatant can be decanted from the cell pellet through an upper port without disturbing the pellet, obviating the need for careful supernatant removal procedures. Moreover, the tube design is such that even "lighter" cells present in the upper portion of the cell pellet are retained during the decantation process.
This tube and process provide the advantages of (i) a closed system for sterile manipulation of transfer of materials into and out of the tube, (ii) a design that allows for thorough decantation of the cell supernatant by inversion of the tube, without appreciable or differential loss of the cells at the top of the pellet. This latter feature of the invention facilitates high yield recovery of rare cells that might otherwise be lost or at least severely depleted during the washing process.
These and other features of the invention are described in the sections which follow.